The overall objective of the research proposed in this grant application is to determine the molecular pathways that act to initiate and complete T cell commitment. Recently we have isolated E2A-deficient hematopoietic progenitor cells that can be grown long term in vitro. Reconstitution of lethally irradiated mice with E2A-deficient hematopoietic progenitor cells leads to the development of T, NK, dendritic, myeloid and erythroid cell lineages, indicating that these cells are pluripotent. E2A deficient hematopoietic progenitor cells simultaneously express low levels of transcripts that encode for transcriptional regulators that contribute to the development of various hematopoietic cell lineages. These include EBF, Pax-5, TCF-1 andGATA-3. Enforced expression of E47 into E2A deficient hematopoietic progenitor cells leads to the activation of B-lineage specific gene expression whereas the transcription of factors involved in the commitment and development of alternative lineages is repressed. To determine how Notch signaling and E-protein activity regulate T lineage commitment, we have begun to examine how the combined activities of E-proteins and Notch signaling regulate down-stream target gene expression. Our preliminary studies show that both E47 and Notch-ICN directly activate HES1 gene expression in uncommitted E2A-deficient hematopoietic progenitor cells. Additionally, GATA-3 and TCF-1 gene expression is, albeit indirectly, regulated by Notch-ICN. In this grant application, we propose to continue these studies. We would determine how Notch signaling and E-proteins act to promote T-lineage commitment. We would identify target genes and enhancer elements that are regulated by Notch-ICN and E-proteins. We would examine how E-proteins and Notch mediated signaling act in concert to induce HES1 gene expression. We would examine the contributions of HES-1 and GATA-3 in promoting T-lineage maturation. We would employ both in vitro and in vivo approaches to clarify the role of Notch-ICN and E-proteins in T-lineage commitment. Overall the dissection of the roles of Notch signaling and transcriptional regulators should clarify the mechanisms by which T cell commitment is initiated and completed.